The present invention relates to polyketides and the polyketide synthase (PKS) enzymes that produce them. The invention also relates generally to genes encoding PKS enzymes and to recombinant host cells containing such genes and in which expression of such genes leads to the production of polyketides. Thus, the invention relates to the fields of chemistry, molecular biology, and agricultural, medical, and veterinary technology.
Polyketides are a class of compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. Polyketides are biologically active molecules with a wide variety of structures, and the class encompasses numerous compounds with diverse activities. Tetracycline, erythromycin, epothilone, FK-506, FK-520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of polyketides. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds.
The biosynthetic diversity of polyketides is generated by repetitive condensations of simple monomers by PKSs that mimic fatty acid synthases but are capable of additional processing reactions (see Carreras et al., 1997, Topic in Current Chemistry, Springer-Verlag, Berlin; and Staunton et al., 1997, Chem. Rev. 97: 2611-2629, each of which is incorporated herein by reference). For instance, the deoxyerythronolide-B (DEBS-B) synthase catalyzes the chain extension of a primer with several methylmalonyl coenzyme A (MeMalCoA) extender units to produce the erythromycin core (see Khosla, 1997, Chem. Rev. 97: 2577-2590, and Hopwood, 1997, Chem. Rev. 97: 2465-2495, each of which is incorporated herein by reference).
The cloning, analysis, and recombinant DNA technology of genes that encode PKS enzymes allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters. See, e.g., PCT publication Nos. WO 93/13663; 95/08548; 96/40968; 97/02358; 98/27203; and 98/49315; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146; 5,830,750; and 5,843,718; and Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.
PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between acylthioester building blocks. The building blocks used to form complex polyketides are typically acylthioesters, such as acetyl, butyryl, propionyl, malonyl, hydroxymalonyl, methylmalonyl, and ethylmalonyl CoA. Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis of the polyketide synthesized. These two major types of PKS enzymes are commonly referred to as Type I or xe2x80x9cmodularxe2x80x9d and Type II xe2x80x9citerativexe2x80x9d PKS enzymes.
In the Type I or modular PKS enzyme group, a set of separate catalytic active sites (each active site is termed a xe2x80x9cdomainxe2x80x9d, and a set thereof is termed a xe2x80x9cmodulexe2x80x9d) exists for each cycle of carbon chain elongation and modification in the polyketide synthesis pathway. The typical modular PKS is composed of several large polypeptides, which can be segregated from amino to carboxy terminii into a loading module, multiple extender modules, and a releasing (or thioesterase) domain. The PKS enzyme known as 6-deoxyerythronolide B synthase (DEBS) is a typical Type I PKS. In DEBS, there is a loading module, six extender modules, and a thioesterase (TE) domain. The loading module, six extender modules, and TE of DEBS are present on three separate proteins (designated DEBS-1, DEBS-2, and DEBS-3, with two extender modules per protein). Each of the DEBS polypeptides is encoded by a separate open reading frame (ORF) or gene; these genes are known as eryAI, eryAII, and eryAIII. See Caffrey et al., 1992, FEBS Letters 304: 205, and U.S. Pat. No. 5,824,513, each of which is incorporated herein by reference. (See FIG. 2.) There is considerable interest in the genetic and chemical reprogramming of modular PKSs (see, e.g., Khosla, 1997, Chem. Rev. 97: 2577-2590, and Staunton et al., 1997, Chem. Rev. 2611-2629, each of which is incorporated herein by reference).
Generally, the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module. The loading module of DEBS consists of an acyltransferase (AT) domain and an acyl carrier protein (ACP) domain. Another type of loading module utilizes an inactivated KS, an AT, and an ACP. This inactivated KS is in some instances called KSQ, where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of the active site cysteine required for ketosynthase activity. In other PKS enzymes, including the FK-520 PKS, the loading module incorporates an unusual starter unit and is composed of a CoA ligase activity domain. In any event, the loading module recognizes a particular acyl-CoA (usually acetyl or propionyl but sometimes butyryl) and transfers it as a thiol ester to the ACP of the loading module.
The AT on each of the extender modules recognizes a particular extender-CoA (malonyl or alpha-substituted malonyl, i.e., methylmalonyl, ethylmalonyl, and carboxylglycolyl) and transfers it to the ACP of that extender module to form a thioester. Each extender module is responsible for accepting a compound from a prior module, binding a building block, attaching the building block to the compound from the prior module, optionally performing one or more additional functions, and transferring the resulting compound to the next module.
Each extender module of a modular PKS contains a ketosynthase (KS), AT, ACP, and zero, one, two, or three enzymes that modify the beta-carbon of the growing polyketide chain. A typical (non-loading) minimal Type I PKS extender module is exemplified by extender module three of DEBS, which contains a KS domain, an AT domain, and an ACP domain. These three domains are sufficient to activate a 2-carbon extender unit and attach it to the growing polyketide molecule. The next extender module, in turn, is responsible for attaching the next building block and transferring the growing compound to the next extender module until synthesis is complete.
Once the PKS is primed with acyl- and malonyl-ACPs, the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, extender module one possesses an acyl-KS adjacent to a malonyl (or substituted malonyl) ACP. The acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbonxe2x80x94carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading building block (elongation or extension).
The polyketide chain, growing by two carbons each extender module, is sequentially passed as covalently bound thiol esters from extender module to extender module, in an assembly line-like process. The carbon chain produced by this process alone would possess a ketone at every other carbon atom, producing a polyketone, from which the name polyketide arises. Most commonly, however, additional enzymatic activities modify the beta keto group of each two carbon unit just after it has been added to the growing polyketide chain but before it is transferred to the next module.
Thus, in addition to the minimal module containing KS, AT, and ACP domains necessary to form the carbonxe2x80x94carbon bond and as noted above, modules may contain a ketoreductase (KR) domain that reduces the keto group to an alcohol. Modules may also contain a KR domain plus a dehydratase (DH) domain that dehydrates the alcohol to a double bond. Modules may also contain a KR domain, a DH domain, and an enoylreductase (ER) domain that converts the double bond product to a saturated single bond using the beta carbon as a methylene function. An extender module can also contain other enzymatic activities, such as, for example, a methylase or dimethylase activity.
After traversing the final extender module, the polyketide encounters a releasing domain that cleaves the polyketide from the PKS and typically cyclizes the polyketide. For example, final synthesis of 6-dEB is regulated by a TE domain located at the end of extender module six. In the synthesis of 6-dEB, the TE domain catalyzes cyclization of the macrolide ring by formation of an ester linkage. In FK-506, FK-520, rapamycin, and similar polyketides, the ester linkage formed by the TE activity is replaced by a linkage formed by incorporation of a pipecolate acid residue. The enzymatic activity that catalyzes this incorporation for the rapamycin enzyme is known as RapP, encoded by the rapP gene. The polyketide can be modified further by tailoring enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule. For example, 6-dEB is hydroxylated at C6 and C12 and glycosylated at C3 and C5 in the synthesis of erythromycin A.
In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker or xe2x80x9cscaffoldxe2x80x9d-encoding regions. These scaffold regions encode amino acid sequences that space the domains at the appropriate distances and in the correct order. Thus, the linker regions of a PKS protein collectively can be considered to encode a scaffold into which the various domains (and thus modules) are placed in a particular order and spatial arrangement. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the DNA level) between PKS enzymes by various available methodologies. Thus, there is considerable flexibility in the design of new PKS enzymes with the result that known polyketides can be produced more effectively, and novel polyketides useful as pharmaceuticals or for other purposes can be made.
There remains a need for new methods and reagents for making polyketides. In particular, methods for altering the stereochemistry of polyketides by altering the PKS enzymes that produce the polyketides, as well as methods for altering the specificity of AT domains of PKS enzymes, without substantial loss of PKS activity are needed. The present invention helps meet the need for such nucleic acid compounds by providing new methods and reagents for manipulating PKS genes.
In one embodiment, the invention provides a method for altering the specificity of an acyltransferase (AT) domain of a polyketide synthase, which method comprises the step of altering the amino acid sequence of the hypervariable region of the AT domain without changing at least a portion of the remaining AT domain amino acid sequence. In one embodiment, the specificity of the AT domain is changed from one naturally occurring starter or extender unit to another. The correct specificity may, if desired, be assured by altering the hypervariable region to mimic that of an AT of the desired specificity such as those shown in FIG. 1.
In another embodiment, the invention provides a method for changing the specificity of an AT domain of a PKS including changing it from that for the naturally occurring starter or extender unit to a starter or extender unit that is not naturally incorporated, said method comprising the steps of altering the amino acid sequence of the hypervariable region of the AT domain by mutagenesis, and if necessary, assessing the resulting specificity by contacting the PKS comprising the altered AT domain with one or more starter or extender units that are not incorporated into polyketides in nature, and identifying whether the altered AT domain can incorporate the tested starter or extender unit. In related embodiments, the invention provides novel PKS enzymes capable of incorporating starter or extender units not natively incorporated and novel polyketides produced thereby.
In another embodiment, the present invention provides non-native extender substrates for PKS enzymes and methods of making such extender substrates. In one embodiment, these non-native extender substrates are used to make polyketides in a cell-free system and in host cells that do not naturally produce polyketides but which can produce polyketides when provided with a recombinant PKS and the appropriate substrates.
In another embodiment, the invention provides a method for altering the stereochemistry of a polyketide by replacing a KS domain in the PKS that produces the polyketide with a KS domain that provides the desired stereochemistry.
These and other embodiments, modes, and aspects of the invention are described in more detail in the following description, the examples, and claims set forth below.